Cloning of hsa-miR-505 for Overexpression In Breast Cancer Cells
Cancer is an abnormal proliferation of cells that can potentially spread and threaten other tissues. Among different types of cancer, breast carcinoma is one of the leading causes of death among women worldwide. MicroRNAs (miRNAs) are 18-24nt long non-coding RNAs. They are involved in post-transcriptional regulation of gene expression. Drosha which is a ribonuclease enzyme cleaves the primary transcript to produce pre-miRNA which is cleaved by the Dicer ribonuclease to generate the mature miRNA and antisense products. In this project, we would construct a lentiviral construct that would overexpress hsa-mir-505-3p. Sequence of hsa-mir-505-3p was retrieved from miRBase. The mature miRNA would be cloned into pLKO.1 TRC cloning vector. For cloning, the protocol recommended by Addgene would be followed with few modifications. The oligos were designed by replacing the XhoI loop sequence with PstI sequence. The 58 nt forward and reverse oligos were annealed and phosphorylated following recommendations of a standard protocol (publicly available on the lab website of Kevin Janes of Virginia State University). The vector was gel purified following digestion with AgeI and EcoRI. The annealed and phosphorylated oligos would further be ligated to the digested vector and the ligated product would be transformed into chemically-competent Stbl3 E. coli cells. Ampicillin-resistant colonies would be screened by digestion with BamHI (having a recognition site in the vector backbone) and PstI. Subsequently, the positive clones would be confirmed Sanger sequencing. Eventually, positive clones would be isolated on a larger scale and used for preparation of lentiviral particle.
Keywords: hsa-miR-505, Breast Cancer, Lentiviral Particle, Cloning, RNA Interference, pLKO.1 TRC vector